Controlled local delivery of chemotherapeutic agents for treating solid tumors

ABSTRACT

A method and devices for localized delivery of a chemotherapeutic agent to solid tumors, wherein the agent does not cross the blood-brain barrier and is characterized by poor bioavailability and/or short half-lives in vivo, are described. The devices consist of reservoirs which release drug over an extended period while at the same time preserving the bioactivity and bioavailability of the agent. In the most preferred embodiment, the device consists of biodegradable polymeric matrixes, although reservoirs can also be formulated from non-biodegradable polymers or reservoirs connected to implanted infusion pumps. The devices are implanted within or immediately adjacent the tumors to be treated or the site where they have been surgically removed.

The U.S. government has rights in this invention by virtue of a grantfrom the National Cancer Institute, cooperative agreement number U01 CA52857; and N.I.H. grant numbers U01 CA52857 to Henry Brem and Robert S.Langer and T32 CA09574.

BACKGROUND OF THE INVENTION

This application is a 371 of US/PCT95/09805, filed Aug. 02, 1995.

This invention is in the field of localized delivery of chemotherapeuticagents to solid tumors.

One-third of all individuals in the United States alone will developcancer. Although the five-year survival rate has risen dramatically tonearly fifty percent as a result of progress in early diagnosis and thetherapy, cancer still remains second only to cardiac disease as a causeof death in the United States. Twenty percent of Americans die fromcancer, half due to lung, breast, and colon-rectal cancer.

Designing effective treatments for patients with cancer has representeda major challenge. The current regimen of surgical resection, externalbeam radiation therapy, and/or systemic chemotherapy has been partiallysuccessful in some kinds of malignancies, but has not producedsatisfactory results in others. In some malignancies, such as brainmalignancies, this regimen produces a median survival of less than oneyear. For example, 90% of resected malignant gliomas recur within twocentimeters of the original tumor site within one year.

Though effective in some kinds of cancers, the use of systemicchemotherapy has had minor success in the treatment of cancer of thecolon-rectum, esophagus, liver, pancreas, and kidney and melanoma. Amajor problem with systemic chemotherapy for the treatment of thesetypes of cancer is that the systemic doses required to achieve controlof tumor growth frequently result in unacceptable systemic toxicity.Efforts to improve delivery of chemotherapeutic agents to the tumor sitehave resulted in advances in organ-directed chemotherapy, as bycontinuous systemic infusion, for example. However, continuous infusionsof anticancer drugs generally have not shown a clear benefit over pulseor short-term infusions. Implantable elastomer access ports withself-sealing silicone diaphragms have also been tried for continuousinfusion, but extravasation remains a problem. Portable infusion pumpsare now available as delivery devices and are being evaluated forefficacy. (See Harrison's Principles of Internal Medicine 431-446, E.Braunwald et al., ed., McGraw-Hill Book Co. (1987) for a generalreview).

In the brain, the design and development of effective anti-tumor agentsfor treatment of patients with malignant neoplasms of the centralnervous system have been influenced by two major factors: 1) theblood-brain barrier provides an anatomic obstruction, limiting access ofdrugs to these tumors; and 2) the drugs given at high systemic levelsare generally cytotoxic. Efforts to improve drug delivery to the tumorbed in the brain have included transient osmotic disruption of the bloodbrain barrier, cerebrospinal fluid perfusion, and-direct infusion into abrain tumor using catheters. Each technique has had significantlimitations. Disruption of the blood brain barrier increased the uptakeof hydrophilic substances into normal brain, but did not significantlyincrease substance transfer into the tumor. Only small fractions ofagents administered into the cerebrospinal fluid actually penetratedinto the brain parenchyma. Drugs that have been used to treat tumors byinfusion have been inadequate, did not diffuse an adequate distance fromthe site of infusion, or could not be maintained at sufficientconcentration to allow a sustained diffusion gradient. The use ofcatheters has been complicated by high rates of infection, obstruction,and malfunction due to clogging. See T. Tomita, "Interstitialchemotherapy for brain tumors: review" J. Neuro-Oncology 10:57-74(1991).

Controlled release biocompatible polymers for local drug delivery havebeen utilized for contraception, insulin therapy, glaucoma treatment,asthma therapy, prevention of dental caries, and certain types of cancerchemotherapy. (Langer, R., and D. Wise, eds, Medical Applications ofControlled Release, Vol. I and II, Boca Raton, CRC Press (1986)) Braintumors have been particularly refractory to chemotherapy. One of thechief reasons is the restriction imposed by the blood-brain barrier.Agents that appear active against certain brain tumors, such as gliomas,in vitro may fail clinical trials because insufficient drug penetratesthe tumor. Although the blood-brain barrier is disrupted at the core ofa tumor, it is largely intact at the periphery where cells activelyengaged in invasion are located. Experimental intratumoral regimensinclude infusing or implanting therapeutic agents within the tumor bedfollowing surgical resection, as described by Tamita, T, J. Neuro-Oncol.10:57-74 (1991).

Delivery of a low molecular weight, lipid soluble chemotherapeutic,1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), in a polymer matriximplanted directly adjacent to brain tumors has some efficacy, asreported by Brem, et al., J. Neurosurg, 74:441-446 (1991); Brem, et al.,Eur. J. Pharm. Biopharm. 39(1):2-7 (1993); and Brem, et al.,"Intraoperative Chemotherapy using biodegradable polymers: Safety andEffectiveness for Recurrent Glioma Evaluated by a Prospective,Multi-Institutional Placebo-Controlled Clinical Trial" Proc. Amer. Soc.Clin. Oncology May 17, 1994. Polymer-mediated delivery of BCNU wassuperior to systemic delivery in extending survival of animals withintracranial 9L gliosarcoma and has shown some efficacious results inclinical trials. However, BCNU is a low molecular weight drug, crossesthe blood-barrier and had previously been demonstrated to have someefficacy when administered systemically.

Unfortunately, the predictability of the efficacy of chemotherapeuticagents remains low. Drugs that look effective when administeredsystemically to animals may not be effective when administeredsystemically to humans due to physiological differences andbioavailability problems, and drugs that are effective systemically maynot be effective when administered locally.

For example, one promising chemotherapeutic, camptothecin, a naturallyoccurring alkaloid isolate from Camptotheca acuminata, a tree indigenousto China, which exerts its pharmacological effects by irreversiblyinhibiting topoisomerase I, an enzyme intimately involved in DNAreplication, has been shown to have strong cytotoxic anti-tumor activityagainst a variety of experimental tumors in vitro, such as the L1210 andrat Walker 256 carcinosarcoma (Venditti, J. M., and B. J. Abbott,Lloydia 30:332-348 (1967); Moertel, C. G., et al., Cancer Chemother.Rep. 56(l):95-101 (1972)). Phase I and II clinical trials ofcamptothecin in human patients with melanoma and advancedgastrointestinal carcinoma, however, have shown unexpectedly severesystemic toxicity with poor tumoral responses, and clinicalinvestigation therefore halted. (Gottlieb, J. A., and J. K. Luce, CancerChemother. Rep. 56(1):103-105 (1972); Moertel, C. G., et. al., CancerChemother. Rep. 56(1):95-101 (1972); Muggia, F. M., et al., CancerChemother. Rep. 56(4):515-521 (1972)). Further pharmacologicalevaluation by Gottlieb, et al., Cancer Chemother. Rep. 54(6):461-470(1970) and Slichenmyer, et al., J. Clin. Pharmacol. 30:770-788 (1990),revealed that the sodium salt formulation of camptothecin was stronglyprotein-bound and required conversion to a lactone structure foractivity. The alkaloid, a 4-ethyl-4-hydroxy-1H-pyrano- 3',4':6,7!indolizino 1,2-b! quinoline-3,14(4H,12H)-dione, having a molecularweight of 348, is not only water insoluble, but even crystallizes out ofacetonitrile-methanol, and does not form stable salts with acids. Thepoor bioavailability may explain the lack of in vivo efficacy.

Many other chemotherapeutics which are efficacious when administeredsystemically must be delivered at very high dosages in order to avoidtoxicity due to poor bioavailability. For example, paclitaxel (taxol)has been used systemically with efficacy in treating several humantumors, including ovarian, breast, and non-small cell lung cancer.However, maintenance of sufficient systemic levels of the drug fortreatment of tumors has been associated with severe, in some cases"life-threatening" toxicity, as reported by Sarosy and Reed, J. Nat.Med. Assoc. 85(6):427-431 (1993).

Paclitaxel is a high molecular weight (854), highly lipophilicdeterpenoid isolated from the western yew, Taxus brevifolia, which isinsoluble in water. It is normally administered intravenously bydilution into saline of the drug dissolved or suspended inpolyoxyethylated castor oil. This carrier has been reported to induce ananaphylactic reaction in a number of patients (Sarosy and Reed (1993))so alternative carriers have been proposed, such as a mixed micellarformulation for parenteral administration, described by Alkan-Onyuksel,et al., Pharm. Res. 11(2), 206-212 (1994). There is also extensivenon-renal clearance, with indications that the drug is removed andstored peripherally. Pharmacokinetic evidence from clinical trials(Rowinsky, E. K., et al., Cancer Res. 49:4640-4647 (1989)) and animalstudies (Klecker, R. W., Proc. Am. Assoc. Cancer Res. 43:381 (1993))indicates that paclitaxel penetrates the intact blood-brain barrierpoorly, if at all, and that there is no increased survival from systemicintraperitoneal injections of paclitaxel into rats with intracranialgliomas. Paclitaxel has been administered in a polymeric matrix forinhibition of scar formation in the eye, as reported by Jampel, et al.,Opthalmic Surg. 22,676-680 (1991), but has not been administered locallyto inhibit tumor growth.

It is therefore an object of the present invention to provide achemotherapeutic composition and method of use thereof which providesfor effective long term release of chemotherapeutic agents that are notstable or soluble in aqueous solutions or which have limitedbioavailability in vivo for treatment of solid-tumors.

It is a further object of the present invention to provide a compositionand method of use for the treatment of solid tumors withchemotherapeutic agents that avoids high systemic levels of the agentand associated toxicities.

SUMMARY OF THE INVENTION

A method and devices for localized delivery of a chemotherapeutic agentto solid tumors, wherein the agent does not cross the blood-brainbarrier and is characterized by poor bioavailability and/or shorthalf-lives in vivo, are described. The devices consist of reservoirswhich release drug over an extended time period while at the same timepreserving the bioactivity and bioavailability of the agent. In the mostpreferred embodiment, the device consists of biodegradable polymericmatrixes, although reservoirs can also be formulated fromnon-biodegradable polymers or reservoirs connected to implanted infusionpumps. The devices are implanted within or immediately adjacent thetumors to be treated or the site where they have been surgicallyremoved.

The examples demonstrate the efficacy of paclitaxel, camptothecin andcisplatin delivered in polymeric implants prepared by compressionmolding of biodegradable and non-biodegradable polymers, respectively,against a number of human tumor cell lines, both in vitro and in vivo.The results are highly statistically significant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing paclitaxel cell kill as determined by aclonogenic assay versus the human glioma U373 cell line with 1-h (LD₉₀=280 nM, open squares), 24-h (LD₉₀ =29 nM, dark circles), and continuous6- to 8- day exposure (LD₉₀ =7.2 nM, open circle) to the drug, shown ascolony count (% of control) log concentration of paclitaxel in nM.

FIG. 2 is a graph of the cumulative percent release over time (hours)for PCPP-SA (20:80) polymer discs (10 mg) loaded with 20% (diamond), 30(triangle) or 40% (square) by weight paclitaxel.

FIG. 3 is a graph of percent survival over time in days (Kaplan-Meiersurvival curves) in rats receiving an intracranial 9L gliosarcoma tumorimplant on day 0 and were treated on day 5 with an intratumoral implantconsisting of a 10-mg disc of PCPP-SA (20:80) loaded with 20, 30 or 40%paclitaxel by weight. Control animal received a 10 mg PCPP-SA (20:80)implant with no loaded drug.

FIG. 4 is a graph of the in vitro cumulative percent release over timein days from 10 mg ethylene vinyl acetate (EVAc) polymer implantsformulated with 20 (triangle), 40 (square), and 50 (circle) percentcamptothecin by weight. Each point represents the mean of threemeasurements.

FIG. 5 is graph of percent survival over time in days (Kaplan-Meiersurvival curves) comparing systemic delivery of camptothecin with localdelivery from EVAc polymers. Rats received an intracranial 9Lgliosarcoma implant on day 0 and treatment was initiated on day 5.Control animals and those treated with i.p. camptothecin received a 9.0mg EVAc polymer implant with no loaded drug. Systemic camptothecin at 20or 40 mg/kg/day was administered i.p. over four days, beginning on day5. The camptothecin polymer group received a 9.2 mg intratumoral implantof EVAc loaded with 50% camptothecin by weight.

DETAILED DESCRIPTION OF THE INVENTION

One method of extending the duration of exposure of a tumor to a drug isto deliver the drug interstitially to the tumor. Controlled-infusionpumps and biodegradable polymer devices are currently being developed todeliver drugs in such a sustained fashion to tumors of the centralnervous system. Interstitial delivery minimizes the systemic drug levelsand side effects of an agent. Delivering chemotherapeutic drugs locallyto a tumor is an effective method of prolonging tumor exposure to thedrug while minimizing the drug's dose-limiting systemic side effects,such as neutropenia.

Interstitial drug delivery also bypasses the limitations of theblood-brain barrier. Presently, it is unclear how well some drugs suchas paclitaxel cross the blood-brain barrier.

As described herein, a composition is formulated of a chemotherapeuticagent which is not water soluble and has poor bioavailability in vivowhich is encapsulated into a biocompatible polymeric matrix, mostpreferably biodegradable, for use in treatment of solid tumors. Theagent is released by diffusion and/or degradation over a therapeuticallyeffective time, usually eight hours to five years, preferably one weekto one year.

Polymeric Formulations

The ideal polymeric matrix would combine the characteristics ofhydrophobicity, stability, organic solubility, low melting point, andsuitable degradation profile. The polymer must be hydrophobic so that itretains its integrity for a suitable period of time when placed in anaqueous environment, such as the body, and be stable enough to be storedfor an extended period before use. The ideal polymer must also bestrong, yet flexible enough so that it does not crumble or fragmentduring use.

Biocompatible polymers can be categorized as biodegradable andnon-biodegradable. Biodegradable polymers degrade in vivo as a functionof chemical composition, method of manufacture, and implant structure.Synthetic and natural polymers can be used although synthetic polymersare preferred due to more uniform and reproducible degradation and otherphysical properties. Examples of synthetic polymers includepolyanhydrides, polyhydroxyacids such as polylactic acid, polyglycolicacids and copolymers thereof, polyesters, polyamides, polyorthoesters,and some polyphosphazenes. Examples of naturally occurring polymersinclude proteins and polysaccharides such as collagen, hyaluronic acid,albumin and gelatin. Drug can be encapsulated within, throughout, and/oron the surface of the implant. Drug is released by diffusion,degradation of the polymer, or a combination thereof. There are twogeneral classes of biodegradable polymers: those degrading by bulkerosion and those degrading by surface erosion. The latter polymers arepreferred where more linear release is required. The time of release canbe manipulated by altering chemical composition; for example, byincreasing the amount of an aromatic monomer such as p-carboxyphenoxypropane (CPP) which is copolymerized with a monomer such as sebacic acid(SA). A particularly preferred polymer is CPP-SA (20:80).

Use of polyanhydrides in controlled delivery devices has been reportedby Leong, et al., J. Med. Biomed. Mater. Res. 19, 941 (1985); J. Med.Biomed. Mater. Res. 20, 51 (1986); and Rosen, et al., Biomaterials 4,131 (1983). The release and physical properties required for processinginto implants are largely determined by the hydrophobicity and molecularweight, with higher molecular weight polymers having more desirablephysical properties. Aromatic polyanhydrides exhibit near zero order(linear) erosion and release kinetics, but have very slow degradationrates. For example, it was estimated that it would take a deliverydevice prepared from p-CPP more than three years to completely degradein vivo. Polymers prepared from linear aliphatic diacids are hydrophilicsolids that degrade by bulk erosion, resulting in a rapid release of thedrug from the polymeric matrix. Further, anhydride homopolymers based onaromatic or linear aliphatic dicarboxylic acids are highly crystallineand have poor film forming properties. Aromatic polyanhydrides also havehigh melting points and low solubility in organic solvents.Copolymerizing the linear aliphatic diacids with aromatic diacids, toform, for example, the copolymer of poly 1,3-(bis(p-carbophenoxy)propaneanhydride (p-CPP) (an aromatic polyanhydride) with sebacic acid (acopolymer of an aromatic diacid and an aliphatic diacid), can be used toobtain polymers having appropriate degradation times. As described inU.S. Pat. No. 4,757,128 to Domb and Langer, high molecular weightcopolymers of aliphatic dicarboxylic acids with aromatic diacids areless crystalline than aromatic or linear aliphatic polyanhydrides, andthey form flexible films. U.S. Patents that describe the use ofpolyanhydrides for controlled delivery of substances include U.S. Pat.No. 4,857,311 to Domb and Langer, U.S. Pat. No. 4,888,176 to Langer, etal., and U.S. Pat. No. 4,789,724 to Domb and Langer.

Other polymers such as polylactic acid, polyglycolic acid, andcopolymers thereof have been commercially available as suture materialsfor a number of years and can be readily formed into devices for drugdelivery.

Non-biodegradable polymers remain intact in vivo for extended periods oftime (years). Drug loaded into the non-biodegradable polymer matrix isreleased by diffusion through the polymer's micropore lattice in asustained and predictable fashion, which can be tailored to provide arapid or a slower release rate by altering the percent drug loading,porosity of the matrix, and implant structure. Ethylene-vinyl acetatecopolymer (EVAc) is an example of a nonbiodegradable polymer that hasbeen used as a local delivery system for proteins and othermacromolecules, as reported by Langer, R., and J. Folkman, Nature(London) 263:797-799 (1976). Others include polyurethanes,polyacrylonitriles, and some polyphosphazenes.

Compounds to be encapsulated

Chemotherapeutic Agents

A variety of different chemotherapeutic agents can be incorporated intothe polymeric matrix. In general, drugs will be added to between 10 and50% (w/w), although the optimum can vary widely depending on the drug,from 1% to 90%.

Table 1 is a summary of studies on intracranial local drug delivery inrat glioma models, as described in the following examples.

                                      TABLE 1                                     __________________________________________________________________________    Summary of intracranial local drug delivery.                                                             Median survival in days                                                       controls                                                                            R.sub.x group                                                                      % increase                              Drug   Tumor   polymer                                                                             R.sub.x day                                                                         (n)   (n)  survival                                                                              p value                         __________________________________________________________________________    campto-                                                                              9L      EVAc  5     19    >120 >530%   <0.001                          thecin (2 × 2 × 1 mm.sup.3)                                                      (50% load)                                                            9L      p(CPP:SA)                                                                           5     24    43   79%     N.A.                                   (2 × 2 × 1 mm.sup.3)                                                      (50% load)  (n = 10)                                                                            (n = 10)                                     BCNU   9L      EVAc  4     10.9  80   634%    <0.05                                  (2 × 2 × 1 mm.sup.3)                                                      (30% load)  (n = 12)                                                                            (n = 12)                                            9L      p(CPP:SA)                                                                           4     11.6  62.3 437%    <0.05                                  (2 × 2 × 1 mm.sup.3)                                                      (30% load)  (n = 12)                                                                            (n = 12)                                     4-HC   9L      p(FAD:SA)                                                                           3     14    77   450%    <0.005                                 (1 × 1 × 1 mm.sup.3)                                                      (20% load)  (n = 10)                                                                            (n = 10)                                            9L      p(CPP:SA)                                                                           5     14    44.5 218%                                           (1 × 1 × 1 mm.sup.3)                                                      (20% load)  (n = 8)                                                                             (n = 8)                                      minocy-                                                                              9L      EVAc  0     13    69   430%    <0.001                          cline  (1 × 1 × 1 mm.sup.3)                                                      (50% load)  (n = 10)                                                                            (n = 10)                                            9L      EVAc  5     13    13   0       NS                                     (1 × 1 × 1 mm.sup.3)                                                      (50% load)  (n = 17)                                                                            (n = 8)                                             9L      EVAc  5     --    --   43%     <0.002                                 (1 × 1 × 1 mm.sup.3)                                                      (50% load)                                                                          (+rsxn)                                                                             (n = 18)                                                                            (n = 17)                                            9L      EVAc  5     --    --   93% > ip BCNU                                                                         <0.002                                 (1 × 1 × 1 mm.sup.3)                                                      (50% load)                                                                          (+ipBCNU)                                                                           (n = 17)                                                                            (n = 23)                                                                           330% > no Rx                                                                          <0.001                          Carbo- F98 inj.                                                                              p(FAD:SA)                                                                           5     16    52   225%    <0.001                          platin (1 × 10.sup.5 cells)                                                            (5% load)   (n = 18)                                                                            (n = 18)                                     Taxol  9L      p(CPP:SA)                                                                           5     19.5  61.5 215%    <0.001                                 (1 × 1 × 1 mm.sup.3)                                                      (20% load)  (n = 8)                                                                             (n = 8)                                      Methotrexate                                                                         9L      P(FAD:SA)                                                                           5     18.6  23.0 60%                                     & dextran mix                                                                        (2 × 2 × 1 mm.sup.3)                                                      (.01% load) (n = 8)                                                                             (n = 8)                                      __________________________________________________________________________

The preferred chemotherapeutic agents are camptothecin and paclitaxel,which are insoluble in water, relatively insoluble in lipid (compared,for example, to BCNU), high molecular weight (i.e., of a molecularweight not normally crossing the blood brain barrier), exhibit rapidnon-renal clearance in vivo, and have substantial systemic toxicity, andtheir functionally effective derivatives. As used herein, paclitaxelrefers to paclitaxel and functionally equivalent derivatives thereof,and camptothecin refers to camptothecin and functionally equivalentderivatives thereof. Other chemotherapeutic agents that may be usefulinclude the platinum based chemotherapeutic agents, carboplatinum andcisplatinum, alone or in combination with other chemotherapeutic agents.

The preferred weight percent range of drug in polymer is from one to 90%and the preferred time of degradation is between one week and one year,for both paclitaxel and camptothecin. Dosages must be optimizeddepending on the size of the implant, the location and size of the tumorto be treated, and the period over which drug is to be delivered. Thesecalculations are routine for those skilled in the art of administeringchemotherapy to tumor patients. In general, the effective dosage of achemotherapeutic agent which is administered locally by extended releasewill be significantly less than the dosage for the same drugadministered for shorter periods of time. For example, as shown in FIG.1, described in Example 1, and Table 3, the LD₉₀ for paclitaxeladministered in a one hour infusion is 280 nM; for paclitaxel in a 24hour infusion it is 29 nM; for continuous extended release it is 7.2 nM.

                  TABLE 2                                                         ______________________________________                                        In vitro efficacy of Camptothecin and                                         Paclitaxel on several cell lines.                                             Camptothecin  Range LD.sub.90 1 h                                                                        LD.sub.90 continuous                               Cell line     exposure (μM)                                                                           exposure (μM)                                   ______________________________________                                        F98                                                                           9L                                                                            U87           0.1 to 1.40  0.026 to 0.10                                      U373                                                                          JH1                                                                           Paclitaxel    Range LD.sub.90 1 h                                                                        LD.sub.90 continuous                               Cell line     exposure (μM)                                                                           exposure (μM)                                   9L                                                                            F98                                                                           U373          0.280 to 0.890                                                                             0.0072 to 0.042                                    H80                                                                           U87                                                                           D324                                                                          ______________________________________                                    

Adriamycin (doxorubicin) is another chemotherapeutic which can beutilized. Gliomas are highly sensitive to this drug in vitro, there issignificant dose related cardiac toxicity, and there is synergy withtumor vaccines and immune based therapy. For incorporation intopolymers, the drug is soluble in water, methanol, and aqueous alcohols.It is insoluble in acetone, benzene, chloroform, ethyl ether, andpetroleum ether. An ideal release profile would have extended releaseover a period of at least one month. The dosage based on LD₉₀ for gliomalines ranges from 10 to 100 ng/ml.

Combinations with other biologically active compounds

These chemotherapeutic agents can also be administered in combinationwith each other or other chemotherapeutic agents, including radiationtherapy. Examples of other chemotherapeutic agents include cytotoxicagents such as ternozolamide, platinum drugs such as cisplatin,differentiating agents such as butyrate derivatives, transforming growthfactor such as factor-alpha-Pseudomonas exotoxin fusion protein, andantibodies to tumor antigens, especially glioma antigens, such asmonoclonal antibody 81C6.

Therapeutic immune responses can be modified by generation andenhancement of a systemic inflammatory response directed against a tumorand enhancement of a local inflammatory response to the tumor.Granulocyte-macrophage colony stimulating factor (GM-CSF) is an exampleof a cytokine systemically activating cytotoxic T lymphocytes (CTL)which has been shown to lead to the elimination of tumor cells in apotent and specific manner, by stimulating the growth and activity ofseveral myeloid cells and playing a critical role in the migration anddevelopment of professional antigen present cells such as dendriticcells. Tumor specific CTL induction and systemic protection from tumorchallenge can be generated by the subcutaneous injection of irradiatedtumor cells genetically modified to produce the cytokinegranulocyte-macrophage colony stimulating factor (GM-CSF). In oneembodiment, killed tumor cells are transduced with the gene encodingGM-CSF and administered as a vaccine to stimulate CTL activation. Thiscan be done prior to or in combination with implantation or localdelivery of the chemotherapeutic agents. Other cytokines such asinterleukin 2 (IL-2), tumor necrosis factor (TNF) and IL-4, as well asIL-5, IL-6 and gamma interferon (although not as well), act locally tostimulate tumor responses. IL-2 induces a local inflammatory responseleading to activation of both helper and cytotoxic subsets of T cells.IL-4 has broad immunoregulatory properties. TNF-α has a diverse range ofbiological properties including generation of a number of cytokines suchas IL-6, IL-8, GM-CSF, and G-CSF, as well as the generation ofhemorrhagic necrosis in established tumors. These are highly effectiveif administered in the polymeric matrix with the chemotherapeutic drugor in the form of transduced cells expressing IL-2 which areco-administered to the animal. Other vaccines and immunotoxins are alsowell known to those skilled in the art.

Examples of preferred combinations include combinations of cytotoxicagents or of cytotoxic agent and inhibitors 4-HC and topoisomeraseinhibitors such as camptothecin, 4-HC and BCNU, BCNU and O6-BG, 4-HC andnovobiocin, 4-HC and novobiocin, and 4-HC and BSO; combinations ofcytoxic agents and other agents such as alkylating agents anddifferentiating agents (4HC and phenylbutyrate) and cytoxic agents andbiologics (antibodies, immunotoxins, or growth factor linked toxins),and combinations of chemotherapeutic, cytokine (interleukin) andfumagillin. Other agents that can be incorporated includeantiangiogenesis agents and radiosensitizers, which are known to thoseskilled in the art. For example, paclitaxel is known to be effective asa radiosensitizer.

The same methods described with reference in the literature toincorporation of BCNU, and herein with reference to incorporation ofcamptothecin and paclitaxel, can be used to incorporate these compoundsinto polymeric matrices. See, for example, Domb, et al., Polym. Prepr.32(2):219-220 (1991), reported incorporating the water solublechemotherapeutic agents carboplatin, an analog of cisplatin, and4-hydroperoxycyclophosphamide into a biodegradable polymer matrix fortreating tumors, with promising results in animals.

In variations of these embodiments, it may be desirable to include otherpharmaceutically active compounds, such as antiinflammatories orsteroids which are used to reduce swelling, antibiotics, antivirals, orantibodies. For example, dexamethasone, a synthetic corticosteroid usedsystemically to control cerebral edema, has been incorporated into anon-biodegradable polymer matrix and tested in rat brain in vitro and invivo for efficacy in reversing cerebral edema. Other compounds which canbe included are preservatives, antioxidants, and fillers, coatings orbulking agents which may also be utilized to alter polymeric releaserates.

Additives that are used to alter properties of the polymeric composition

In the preferred embodiment, only polymer and drugs to be released areincorporated into the delivery device, although other biocompatible,preferably biodegradable or metabolizable, materials can be included forprocessing purposes.

Buffers, acids and bases are used to adjust the pH of the composition.Agents to increase the diffusion distance of agents released from theimplanted polymer can also be included.

Fillers are water soluble or insoluble materials incorporated into theformulation to add bulk. Types of fillers include sugars, starches andcelluloses. The amount of filler in the formulation will typically be inthe range of between about 1 and about 90% by weight.

Spheronization enhancers facilitate the production of sphericalimplants. Substances such as zein, microcrystalline cellulose ormicrocrystalline cellulose co-processed with sodium carboxymethylcellulose confer plasticity to the formulation as well as implantstrength and integrity. During spheronization, extrudates that arerigid, but not plastic, result in the formation of dumbbell shapedimplants and/or a high proportion of fines. Extrudates that are plastic,but not rigid, tend to agglomerate and form excessively large implants.A balance between rigidity and plasticity must be maintained. Thepercent of spheronization enhancer in a formulation depends on the otherexcipient characteristics and is typically in the range of 10-90% (w/w).

Disintegrants are substances which, in the presence of liquid, promotethe disruption of the implants. The function of the disintegrant is tocounteract or neutralize the effect of any binding materials used in theformulation. The mechanism of disintegration involves, in large part,moisture absorption and swelling by an insoluble material. Examples ofdisintegrants include croscarmellose sodium and crospovidone which aretypically incorporated into implants in the range of 1-20% of totalimplant weight. In many cases, soluble fillers such as sugars (mannitoland lactose) can also be added to facilitate disintegration of theimplants.

Surfactants may be necessary in implant formulations to enhancewettability of poorly soluble or hydrophobic materials. Surfactants suchas polysorbates or sodium lauryl sulfate are, if necessary, used in lowconcentrations, generally less than 5%.

Binders are adhesive materials that are incorporated in implantformulations to bind powders and maintain implant integrity. Binders maybe added as dry powder or as solution. Sugars and natural and syntheticpolymers may act as binders. Materials added specifically as binders aregenerally included in the range of about 0.5%-15% w/w of the implantformulation. Certain materials, such as microcrystalline cellulose, alsoused as a spheronization enhancer, also have additional bindingproperties.

Various coatings can be applied to modify the properties of theimplants. Three types of coatings are seal, gloss and enteric. The sealcoat prevents excess moisture uptake by the implants during theapplication of aqueous based enteric coatings. The gloss coat improvesthe handling of the finished product. Water-soluble materials such ashydroxypropyl cellulose can be used to seal coat and gloss coatimplants. The seal coat and gloss coat are generally sprayed onto theimplants until an increase in weight between about 0.5% and about 5%,preferably about 1% for seal coat and about 3% for a gloss coat, hasbeen obtained.

Enteric coatings consist of polymers which are insoluble in the low pH(less than 3.0) of the stomach, but are soluble in the elevated pH(greater than 4.0) of the small intestine. Polymers such as Eudragit®,RohmTech, Inc., Malden, Mass., and Aquateric®, FMC Corp., Philadelphia,Penn., can be used and are layered as thin membranes onto the implantsfrom aqueous solution or suspension. The enteric coat is generallysprayed to a weight increase of about one to about 30%, preferably about10 to about 15% and can contain coating adjuvants such as plasticizers,surfactants, separating agents that reduce the tackiness of the implantsduring coating, and coating permeability adjusters. Other types ofcoatings having various dissolution or erosion properties can be used tofurther modify implant behavior. Such coatings are readily known to oneof ordinary skill in the art.

Preparation of Polymeric-Drug Compositions

Controlled release devices are typically prepared in one of severalways. For example, the polymer can be melted, mixed with the substanceto be delivered, and then solidified by cooling. Such melt fabricationprocesses require polymers having a melting point that is below thetemperature at which the substance to be delivered and polymer degradeor become reactive. Alternatively, the device can be prepared by solventcasting, where the polymer is dissolved in a solvent, and the substanceto be delivered dissolved or dispersed in the polymer solution. Thesolvent is then evaporated, leaving the substance in the polymericmatrix. Solvent casting requires that the polymer be soluble in organicsolvents and that the drug to be encapsulated be soluble or dispersiblein the solvent. Similar devices can be made by phase separation oremulsification or even spray drying techniques. In still other methods,a powder of the polymer is mixed with the drug and then compressed toform an implant.

Methods of producing implants also include granulation, extrusion, andspheronization. A dry powder blend is produced including the desiredexcipients and microspheres. The dry powder is granulated with water orother non-solvents for microspheres such as oils and passed through anextruder forming "strings" or "fibers" of wet massed material as itpasses through the extruder screen. The extrudate strings are placed ina spheronizer which forms spherical particles by breakage of the stringsand repeated contact between the particles, the spheronizer walls andthe rotating spheroniter base plate. The implants are dried and screenedto remove aggregates and fines.

These methods can be used to make microimplants (microparticles,microspheres, and microcapsules encapsulating drug to be released),slabs or sheets, films, tubes, and other structures. A preferred formfor infusion or injection is micro-implants.

Administration to Patients

The chemotherapeutic agents described herein or their functionallyequivalent derivatives can be administered alone or in combination with,either before, simultaneously, or subsequent to, treatment using otherchemotherapeutic or radiation therapy or surgery. A preferred embodimentis the local administration, by implantation of a biocompatiblepolymeric matrix loaded with the chemotherapeutic agent, orinjection/infusion of micro-implants, using dosages determined asdescribed herein. The dosages for functionally equivalent derivativescan be extrapolated from the in vitro and in vivo data.

The composition can also be administered locally using an infusion pump,for example, of the type used for delivering insulin or otherchemotherapeutic agents to specific organs or tumors, although thepolymeric devices has clear advantages to the use of a pump, even animplanted pump having a refillable reservoir, particularly in view ofthe effective dosage ranges, which are so significantly less than thosefor systemic administration.

In the preferred method of administration, the polymeric implants areimplanted at the site of a tumor, either following surgical removal orresection or by injection using microparticles less than about 100 to200 microns in diameter injected by means of a catheter or syringe. Ifbiodegradable polymers are used, it is not necessary to remove theimplant following release of the chemotherapeutic.

The polymeric implants can also be combined with other therapeuticmodalities, including radiotherapy, other chemotherapeutic agentsadministered systemically or locally, and immunotherapy.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1

In vitro efficacy of paclitaxel.

Cell culture. Tumor sensitivity to paclitaxel was measured by theclonogenic assay described by Rosenblum, et al., Cancer 41:2305-2314(1978) and Salcman, et al., Neurosurgery 29:526-531 (1991) with ratglioma (9L, F98), human glioma (H80, U87, U373), and humanmedulloblastoma (D324) cell lines. Cells were grown and propagated inminimum essential medium (MEM) supplemented with 10% fetal bovine serum,L-glutamine, penicillin, and streptomycin and incubated at 37° C. in anatmosphere containing 5% CO₂. At the start of each assay, 600 tumorcells in 2 ml of medium were plated on Falcon 6-well tissue-cultureplates (Becton-Dickenson, Lincoln Park, N.J.). After incubating for 24h, the medium was removed from the places and replaced with 2 ml ofmedium containing paclitaxel and 0.1% dimethylsulfoxide (DMSO). Thetreatment solutions were prepared as described by Roytta et al.,Prostate 11:95-106 (1987). The paclitaxel treatment solution was theneither replaced with fresh paclitaxel-free media containing 0.1% DMSOafter 1 or 24 h or was left on the places for the 6- to 8-day incubationperiod. At the end of the incubation period the plates were stained witha solution containing 0.63 g of Coomassie blue (Sigma), 125 ml ofmethanol, 87 ml of H₂ O, and 38 ml of ascetic acid. The colonies on eachplate were counted and the result was expressed as a percentage of thecolonies formed on control plates not exposed to paclitaxel. Platingefficiencies for control plates ranged from 20% to 25%. A range of drugconcentrations was applied to each set of cells. The percentage of cellkill values for the paclitaxel treatments were plotted as a function ofthe drug concentration used. The concentration of drug necessary toproduce 1 log of cell kill (LD₉₀) was interpolated from the graph.Graphs were prepared and analyzed using Cricket Graph v. 1.3.2 (CricketSoftware, Malvern, Penn.). Each determination was done in triplicate.

Chemicals. Paclitaxel for these experiments, which was provided by theNCI (NSC 125973/44), was used without further purification and stored asa bulk solid at 4° C. A 1 mM stock solution of paclitaxel in DMSO wasprepared and kept at -20° C. until thawed for use. F98 glioma cellscarried for five years were initially provided by Dr. Joseph Goodman,Department of Neurosurgery, Ohio State University, Columbus, Ohio. 91glioma cells carried for eight years were originally obtained from Dr.Marvin Barker of the University of California, San Francisco, Calif.D324 (DAOY), described by Jacobsen, et al., J. Neuropathol. Exp. Neurol.44:472-485 (1985) was provided by Dr. Henry Friedman, Division ofPediatric Hematology and Oncology, Department of Pediatrics, DukeUniversity, Durham, N.C. U373, U87 (described by Beckman, et al., Hum.Hered. 21:238-241 (1971), and H80 (U251) (Bullard, et al., J.Neuropathol. Exp. Neurol. 40:410-427 (1981)) were obtained from theAmerican Type Culture Collection, Rockville, Md.

Results

The effects of paclitaxel on colony formation in vitro in the rat glioma(9Lm F98), human glioma (U87, U373, H80), and human medulloblastoma(D324) tumor lines are shown in Table 3. All of the cell lines weresensitive to the drug when exposed continuously to paclitaxel for 6-8days. Log cell kill (LD₉₀) occurred at values ranging between 3.9 (D324)and 4 nM (9L). The human tumor lines were uniformly more susceptible topaclitaxel than were the rat lines. Log cell kill occurred atnanomolecular paclitaxel concentrations for three (U87, U373, H80) ofthe four human lines studied, whereas four to ten times these amountswere required for the rat lines (9L, F98).

                  TABLE 3                                                         ______________________________________                                        LD.sub.90 values for paclitaxel against                                       malignant brain tumor cell lines as                                           determined by clonogenic assay.                                                      Treatment duration LD.sub.90 concentration (nM) for                           9L     F98    U373     H80  U87    D324                                ______________________________________                                        1 h      890             280                                                  24 h     100             29                                                   6-8 days 42       25     7.2    19   9.1    3.9                               ______________________________________                                    

The duration of exposure to the drug significantly affected paclitaxel'spotency in vitro. After exposure of cells to paclitaxel for only 1 h,the LD₉₀ increased by factors of more than for the 9L line and 40 forthe U373 lines as compared with the values recorded for the continuous(6- to 8-day) exposure. Cells exposed to paclitaxel for 24 h gave LD₉₀values between those obtained -for 1-h and continuous exposure. Forexample, for the human U373 line, FIG. 1 shows that the LD₉₀ for 1-hexposure was 280 nM, that for 24-h exposure was 29 nM, and that forcontinuous exposure was 7.2 nM for the human U373 line.

Paclitaxel has previously been shown to be stable in cell-culturemedium, by Ringel, et al., J. Pharmacol. Exp. Ther. 242:692-698 (1987).It equilibrates with its equipotent epimer, 7-epitaxol, but undergoessignificant (less than 10%) hydrolysis to inactive compounds. Thepotency of the paclitaxel solutions should therefore not have diminishedduring the course of the 6- to 8-day incubation.

These results demonstrate that paclitaxel is highly potent in vitroagainst the rat and human brain-tumor cell lines examined. Log cell killoccurred at nanomolecular concentrations of the drug, which isconsistent with the reports of paclitaxel's activities against othermalignancies in vitro. For example, nanomolecular concentrations ofpaclitaxel have been found to be cytotoxic in vitro against ovarian,breast, lung, and prostatic cancer and melanoma, as reported byHanauske, et al., Anticancer Drugs 3:121-124 (1992); Rowinsky, et al.,Cancer Res. 4093-4100 (1988); Roytta, et al., Prostate 11:95-106 (1987).Furthermore, paclitaxel has demonstrated efficacy against each of thesetumors in clinical trials, as reported by Roth, et al., Pr. Annu. Meet.Am. Soc. Clin. Oncol. 11:A598 (1992); Rowinsky, et al., (1990). Braintumors therefore appear to be as sensitive to paclitaxel in vitro asother tumor lines that are currently being treated with paclitaxel inclinical trials. Moreover, cell sensitivity to paclitaxel concentrationincreased significantly with increasing duration of exposure to the drug(from 1 h to 1 week) in vitro. This finding is consistent with previousinvestigators' reports on paclitaxel's action in vitro against othermalignancies. Paclitaxel arrests the cell cycle during the late G₂ or Mphase but does not slow cell progression through the preceding stages ofcell replication, as described by Horwitz, et al., Ann. NY Acad. Sci.466:733-744 (1986). Increasing the duration of exposure to paclitaxelallows more cells in a given sample to enter the cell-cycle phasesduring which paclitaxel is active. With shorter periods of exposure tothe drug, a greater proportion of cells exist entirely outside thepaclitaxel-sensitive G₂ and M phases during the treatment interval.

To maximize the clinical efficacy of paclitaxel, therefore, a drugdelivery protocol that could maintain an elevated concentration of drugfor an extended period would be desirable. To date, the protocolsdeveloped in clinical phase I trials have generally involved a single 1-to 24-h infusion repeated every 2-3 weeks or a 1- to 6-h infusion givenonce a day for 5 days. The elimination half-lives determined in thesestudies indicate that paclitaxel is cleared relatively rapidly with anelimination half-life. t1/2β, of between 1.3 and 8.6 h (Rowinsky, etal., (1988)). Since 93.5% of a drug is eliminated after four half-lives,most of the paclitaxel is dissipated in these regimens at between 5 and26 h after its administration. The results described herein indicate,however, that paclitaxel's potency increases in vitro by a factor of twoto four times when cells are exposed to paclitaxel for more than 24 h.

EXAMPLE 2

Preparation of paclitaxel implant.

Solid paclitaxel, obtained from Napro Biotherapeutics (Boulder, Colo.)or from the National Cancer Institute (Bethesda, Md.), was mixed withpoly bis(p-carboxyphenoxy)propane-sebacic acid! copolymer (PCPP-SA)(20:80) synthesized according to the method of Domb, A. J., and R.Langer (J. Polym. Sci. 25:3373-3386 (1987)), the teachings of which areincorporated herein by reference, to give a mixture containing 0, 20,30, or 40% paclitaxel by weight. The paclitaxel-polymer mixture wasdissolved in methylene chloride (Fluka, Switzerland) to give a 10%solution (w:v). The solvent was evaporated with a nitrogen stream toyield a dry powder. Paclitaxel-polymer discs (10 mg final weight) wereprepared by compression molding 11 mg of the paclitaxel-polymer powderwith a stainless steel mold (internal diameter, 2.5 mm) under lightpressure from a Carver Press at 200 psi. The discs were sterilized underUV light for 45 minutes.

EXAMPLE 3

Demonstration of paclitaxel delivery from a biodegradable matrix intothe surrounding medium in vitro.

The efficiency of the delivery of paclitaxel incorporated into abiodegradable polymer into the surrounding medium was assessed in vitroas follows.

Preparation of polymer discs. Polymer discs were prepared as describedabove except that ³ H-labeled paclitaxel (Atomic Energy Commission,Nuclear Research Center, Beer Sheva, Israel) was used in the polymerpreparation. The ³ H-labeled paclitaxel had a final specific activity of0.019 μCi/mg and was obtained by mixing ³ H-labeled paclitaxel at 6.2Ci/mmol with 100 mg of unlabeled paclitaxel (Napro Biotherapeutics,Boulder, Colo.; or National Cancer Institute, Bethesda, Md.) in methanoland then evaporating the solvent.

Protocol. The paclitaxel-loaded polymer discs were placed in amicroporous polyethylene specimen capsule (8×8 mm internal diameter andheight), which was immersed in 7 ml of 0.1M phosphate buffer, pH 7.4.The apparatus was placed in a 37° C. incubator. The releasing medium wasreplaced at specified times during the 45-day (1000 hour) incubationperiod, and the recovered solutions were analyzed by scintillationcounting and high pressure liquid chromatography (HPLC). HPLC analysisto confirm the release of intact paclitaxel was performed by extracting2 ml of solution with methylene chloride and evaporating to dryness. Theproduct was then redissolved in methanol and injected onto a C_(1a) HPLCcolumn (Licosphere-100RP-18, 5 mm; E Merck, Darmstadt, Germany) as partof a Merck Hitachi system composed of a L-4200 UV-Vis Detector, L-6200intelligent pump, and D-2500 Chromato integrator). The mobile phaseconsisted of methanol:water (70:30) and detection was at 230 nm. Controlsolutions containing 100 mM paclitaxel in methanol were used todetermine the retention time of paclitaxel under these conditions (6.73to 9.04 minutes). Radioactive analysis to quantify the amount ofpaclitaxel release was done by mixing 200 μl of the releasing buffersolution with 4 ml of a scintillation mixture composed of toluene andLumax (Landgraat, The Netherlands) scintillation mixture in a 2:1 volumeratio. This solution was counted on a 1211 Rack β-liquid scintillationcounter (LKB-Wallac OY, Finland). Each measurement represents theaverage of 3 independent countings. At the end of the release period,the amount of drug remaining in the disc was quantified by dissolvingthe polymer remnant in methylene chloride and counting the solution bythe above technique. Release was measured over time from discscontaining 20, 30, and 40% paclitaxel by weight.

Results. The results of the in vitro drug release study are shown inFIG. 2, which illustrates that the delivery of paclitaxel from thebiodegradable matrix into the surrounding physiological medium wasefficient. HPLC confirmed that the paclitaxel released into buffercorresponded with intact paclitaxel. For each of the polymer loadings, aburst of drug release over the first few hours in solution was followedby essentially zero order kinetic release to the end of the experiment.The most efficient release was obtained from the 20% loaded disc, whichreleased 80% of its loaded paclitaxel within the 1000-hour experimentalperiod. The total amount of paclitaxel released from each disc wasapproximately the same, however: 1.6 mg for 20% disc, 1.8 mg for 30%disc, and 2.0 mg for 40% disc.

These results show that paclitaxel is released biphasically from thepolymer, with an initial burst phase followed by a slower constantrelease phase. It is likely that the burst phase corresponds to therapid release of paclitaxel particles embedded within the matrixsurface, while the prolonged release represents slower release ofpaclitaxel from the center of the matrix. The loading of the polymerdoes not seem to correlate directly with the amount of paclitaxelreleased during the experimental period. Although the 40% loaded polymercontained twice as much paclitaxel as the 20% loaded disc, the 40%loaded disc only released 1.25 times as much paclitaxel as the 20%loaded disc after 800 hours in a saline bath. If polymer degradationwere the sole determining factor of paclitaxel release, then one wouldexpect the loading to correlate directly with total drug released. Sincethe correlation appears weakened, another factor must be at leastpartially controlling paclitaxel release from the disc. Most likely, thelow aqueous solubility of paclitaxel limits its uptake into media,despite breakdown of the matrix. Alternatively, the hydrophobicity ofpaclitaxel may inhibit hydrolysis of the polyanhydride matrix. Whilesuch interactions do not preclude the clinical use of this formulation,they do make the pharmacokinetics of the preparation more complex andthe results less predictable when implanted in vivo as compared withtopically applied or systemically administered.

EXAMPLE 4

Demonstration of intracerebral paclitaxel delivery from the polymermatrix in vivo.

The efficiency of the delivery of paclitaxel from the polymer matrixinto surrounding brain tissue and the concentration of active paclitaxelwithin the brain, as measured up to one month after surgical implant,were assessed as follows.

Animals. Male Fischer 344 rats weighing 200-225 g were obtained fromHarlan Sprague-Dawley, Inc. (Indianapolis, Ind.), kept in standardanimal facilities with 4 rats/cage, and given free access to CertifiedRodent Chow No. 5002 (Ralston Purina Co., St. Louis, Mo.) and toBaltimore city water.

Anesthesia. Rats were anesthetized with an intraperitoneal injection of2-4 ml/kg of a stock solution containing ketamine hydrochloride (25mg/ml), xylazine (2.5 mg/ml), and 14.25% ethyl alcohol in normal saline.They were allowed to recover in their cages following all surgicalprocedures.

Euthanasia. Prior to euthanasia, the rats were anesthetized as above.Euthanasia was accomplished with an intracardiac injection of 0.3 ml ofEuthanasia-6 Solution CII™ (Veterinary Laboratories, Inc., Lenexa,Kans.).

Preparation of the paclitaxel loaded polymer. Polymer discs containinglabeled paclitaxel were prepared as above, except that a small amount of³ H-labeled paclitaxel (specific activity, 19.3 Ci/mmol; National CancerInstitute) in toluene was added to the initial solution of polymer andpaclitaxel in methylene chloride. The polymer-paclitaxel mixture wasthen dried in a vacuum desiccator and pressed into discs using a tablevise calibrated to form a pellet.

Paclitaxel-polymer implantation. The procedure for polymer implantationin the rat has been described by Tamargo, R. J., et al. (Cancer Res.53:329-333 (1993)), the teachings of which are incorporated herein byreference. Briefly, the heads of anesthetized rats were shaved andprepared aseptically. The skull was exposed with a midline incision, anda 3-mm burr hole was drilled through the skull 5 mm posterior and 3 mmlateral to the bregma. The dura was incised with a microsurgical knife(Edward Weck and Co., Inc., Research Triangle Park, N.C.), and thepolymer disc was inserted into the brain parenchyma. The wound wasirrigated and closed with surgical clips (Clay Adams, Parsippany, N.J.).

Protocol for intracerebral drug distribution. Twelve rats were givenimplants of 10-mg discs containing 40% paclitaxel by weight and 0.60μCi/mg disc weight. At 3, 9, 17, and 30 days postimplantation, groups of3 rats each were euthanized. The skull was opened and the brain exposed.The polymer disc was removed from the brain in situ. The brain was thenremoved from the skull and snap frozen in heptane over dry ice. Thebrain was sectioned in the midline into implant and contralateralhemispheres. Each hemisphere was sectioned coronally at 2-mm intervalsby using a tissue blade grid consisting of individual tissue bladesarranged in parallel separated by 2-mm metal spacers. Each section wasweighed, dissolved in 15 ml Solvable homogenizing solution (New EnglandNuclear Dupont, Boston, Mass.), and combined with 15 ml Atomlight™scintillation mixture (New England Nuclear Dupont). The brain sampleswere counted on a Beckman liquid scintillation counter. Raw counts werecorrected for quenching and converted to dpm using a linear regressionbased on a series of quenched standards.

To convert dpm/mg tissue to paclitaxel concentration, a secondexperiment was performed. Four rats were given implants of 40% loadedpolymer discs with 0.39 μCi/mg. One rat each was sacrificed at 3, 9, 17,and 30 days. The brain was removed and frozen as above. A 2-mm coronalsection was taken through the site of the polymer implant. The sectionwas minced and extracted with ethanol. The ethanol fraction was dividedin two. The first half was dried in a vacuum desiccator and thenresuspended in 100 μl of ethanol. Samples of this solution were spottedon silica thin layer chromatography plates (Sigma, St. Louis, Mo.). Asolution of nonradioactive paclitaxel in ethanol was also applied to theplates over the ethanol extract. The plate was developed with methylenechloride:methanol (95:5) and exposed in an iodine chamber. The R_(f)value for the paclitaxel was determined and each lane cut into 4sections: A, origin; B, origin to paclitaxel spot; C, paclitaxel spot;and D, paclitaxel spot to solvent front. The chromatography strips werecombined with Atomlight™ mixture and counted in a liquid scintillationcounter. The distribution of labeled paclitaxel across thechromatography plate allowed determination of signal corresponding tointact drug. To determine the efficiency of extraction, the remaininghalf of the original extract was combined with mixture and counted, andthe residual brain tissue was homogenized and counted as above. Thepaclitaxel concentration in ng/mg brain tissue was calculated bymultiplying the percentage of intact paclitaxel by the dpm/mg brain anddividing by the specific activity of paclitaxel present in the polymerdisc.

Results. The results of the intracerebral distribution studies,demonstrating that the implant is capable of producing elevated brainlevels of paclitaxel throughout the rat brain. These results indicatethat paclitaxel penetrates the brain parenchyma at concentrations thatare theoretically tumoricidal in vitro and that paclitaxel concentrationremains elevated intracranially for prolonged duration, extending thetherapeutic period.

Paclitaxel from the implant was distributed widely throughout the ratbrain. Concentrations were 100 to 1000 ng/mg brain tissue within 2 to 3mm of the implant, but only 1 to 10 ng/mg in brain tissue more than 4 mmaway from the implant and throughout the contralateral hemisphere.Paclitaxel concentrations increased slightly during the 30 days,correlating with additional drug released from the polymer disc. Thepercentage of radioactivity in each slice corresponding to intactpaclitaxel as measured by thin layer chromatography did not changeappreciably over the course of the experiment. At each time point,approximately 56±3% (standard error of the mean) of the raw countsrepresented parent drug. Thirteen ±2% represented polar metabolites, and31±3% were tissue bound. The detection limit for the overall assay was0.2 ng/mg brain tissue.

Although paclitaxel concentration fell off sharply from 100 to 1000ng/mg (μM) brain tissue in the implant hemisphere within 1 to 3 mm ofthe implant to 1 to 10 ng/mg (μM) brain tissue at the periphery of therat brain (7 mm) and throughout the contralateral hemisphere, even theselower concentrations were 2 to 3 orders of magnitude higher than the 90%lethal dose concentrations of paclitaxel for several human (U87, U373,H80, D324) and rat (9L, F98) glioma lines in vitro. Given thehydrophobicity of paclitaxel, it is believed that these levels representthe saturation point of paclitaxel within the interstitial milieu.

Notably, paclitaxel concentration remained elevated for at least onemonth after implantation in the rat brain. Based on in vitro data,prolonging exposure to paclitaxel significantly decreases theconcentration of paclitaxel necessary to achieve log cell kill for bothglial and other tumors (Rowinsky, E. K., et al., Cancer Res.48:4093-4100 (1988)). Thus, the polymer implant appears to maximize theantitumor benefit of paclitaxel. Pharmacokinetic studies from Phase Itrials indicate that paclitaxel is cleared relatively quickly from thecirculation with a β-half-life between 1.3 and 8.6 hours (Rowinsky, E.K., et al., J. Matl. Cancer Inst. 82:1247-1259 (1990)). Since 93.5% of adrug is cleared after four half-lives, it is unlikely that systemicadministration could maintain such high intracranial paclitaxelconcentrations for prolonged periods, especially since the blood-brainbarrier limits uptake of the drug.

In comparison, studies examining the intracerebral distribution of thenitrosourea, BCNU, delivered from the PCPP-SA (20:80) polymer in therabbit brain demonstrated that BCNU was initially distributed widelyfrom the polymer, up to 12 mm from the implant site, with an averageconcentration of 8 mM (Grossman, S., et al., J. Neurosurg. 76:640-647(1992)). Preliminary studies have shown that this concentration is twoorders of magnitude higher than the 90k lethal dose for BCNU against ratglioma cells based on in vitro measurements. The amount of brain exposedto BCNU begins to decrease after three days. However, BCNU is onlydetectable within a 40 mm radius from the implant on Day 7 and onlywithin a 3-mm radium by 21 days after implantation. In contrast andsurprisingly, paclitaxel concentration is still constant or risingthroughout the rat brain 30 days after implant. Thus, paclitaxel appearsto be a better candidate than BCNU for sustained interstitialchemotherapy on a pharmacokinetic basis.

EXAMPLE 5

Demonstration of amount of implant toxicity.

The amount of toxicity associated with the paclitaxel loaded polymerimplant in the brain was determined as follows.

Protocol. Animals were obtained, housed, anesthetized, and sacrificed asdescribed above. PCPP-SA (20:80) discs (10 mg) containing 20, 30, and40% paclitaxel by weight were implanted intracranially in rats by thetechnique described above. A group of control rats were given implantsof blank PCPP-SA discs containing no paclitaxel. Rats were examinedtwice daily for signs of neurotoxicity with respect to grooming,response to startle stimulus, and gait. After 60 days, all survivingrats were sacrificed and their brains were removed and fixed informalin. One coronal brain section centered through the polymer implantwas taken for each rat and stained with hematoxylin and eosin.

Results. The results of the implant toxicity studies are shown in Table4.

                  TABLE 4                                                         ______________________________________                                        Toxicity of Intracranial paclitaxel                                           implants in Fischer 344 rats.                                                 Treatment No. of                  Date of death                               postimplant)                                                                            Survivors/total                                                                          Toxicity     (days                                       ______________________________________                                        Control PCPP-SA                                                                         4/4        --*          --                                          20% paclitaxel                                                                          1/4        Ataxia, hemiparesis,                                                                       41,53,53                                                         weight loss, death                                       30% paclitaxe1                                                                          4/4        --                                                       40% paclitaxel                                                                          4/4        --                                                       ______________________________________                                         *no observed clinical toxicity or death.                                 

As shown in Table 4, there was no apparent acute clinical toxicity fromthe implant. All of the rats recovered from the implant surgery and wereindistinguishable from controls in terms of motor activity, response tostimulus, and grooming. Two rats later developed ataxia and,subsequently, hemiplegia contralateral to the implant, weight loss, anddeath. One rat died spontaneously without a prodrome. All of the otherrats remained neurologically intact throughout the experiment.Histological examination of brain tissue sections through thepaclitaxel-polymer implant site showed scattered foci of karyorrhecticnuclei interspersed with areas of normal brain. In addition, there werescattered cytologically atypical cells with large, hyperchromatic,sometimes bilobed, nuclei. These atypical cells were more numerousaround the implant site, but were seen bilaterally. The changes werepresent to varying degrees in the brains of all rats receiving thepaclitaxel-polymer implant, but were absent in rats receiving the blankPCPP-SA disc, indicating that the cytological changes were a result ofthe paclitaxel exposure. There was no quantitative or qualitativedifference in the visible cytological changes occurring in animals withpaclitaxel implants either between groups with different paclitaxelconcentration implants or between animals that exhibited grossneurobehavioral toxicity and those that did not. The degree ofcytological pathology was, therefore, spread evenly among the differentpaclitaxel-polymer preparations.

A minimal amount of clinical and histological toxicity was associatedwith the paclitaxel-polymer implant in the rat brain. Three of the 12rats receiving the implant (20% loaded polymer) without tumor diedduring the 60-day experimental period, while the other rats toleratedthe treatment without any apparent clinical symptoms. Two rats receivingpaclitaxel (20 and 30% loaded polymers) after tumor implantation alsodied without visible tumor, but with the atypia found in all the ratsreceiving the paclitaxel implant. These atypical changes were consistentwith similar cytological alterations produced by a wide variety ofchemotherapeutic agents. The symptoms of overt toxicity appeared late inthe experiment, 30 days or more after implant, indicating that acutetoxicity is not a primary concern. Interestingly, all the rats receivingthe paclitaxel implant had cytological abnormalities visible onmicroscopic examination, and there was no correlation between the degreeof atypia and the presence or severity of clinical toxicity. From thepharmacokinetic measurements of intracerebral drug distributionfollowing implantation, it is apparent that these devices produced highdrug concentrations distributed throughout the rat brain. Theseconcentrations were maintained intracranially, for at least one monthafter implant. It is not surprising, therefore, that the brain itselfwas affected after prolonged exposure to such high paclitaxelconcentrations. Nevertheless, several rats from the tumor-polymerstudies lived 120 days or more after implantation, and two lived for oneyear. Lower doses of paclitaxel could be used in patients, either from aproportionately smaller polymer disc or from a disc with a lowerpercentage loading of paclitaxel. These doses could be better toleratedfor chronic therapy clinically. Other agents administered interstitiallyto the brain have been reported to show toxicity in animal modelswithout measurable toxicity in clinical trials. Appropriate dosages fortreatment of patients can be determined using standard and routinemethods.

EXAMPLE 6

Demonstration of the efficacy of the paclitaxel loaded polymer implantat extending survival in rats bearing intracranial 9L gliosarcoma.

The efficacy of the paclitaxel loaded polymer implant at extendingsurvival in rats bearing intracranial 9L gliosarcoma was measured asfollows.

Tumor. The 9L gliosarcoma was obtained in 1985 from Marvin Barker, BrainTumor Research Center, University of California, San Francisco, Calif.,and maintained subcutaneously in the flank of male Fischer 344 rats. Thetumor was passaged every 2 to 3 weeks. To harvest tumor for passage andintracranial studies, the flank of a rat bearing the 9L gliosarcoma wasshaved and prepared aseptically with 70% ethanol and povidone-iodine. Anincision was made in the flank, and the tumor was removed en bloc andsectioned into 1-mm³ pieces, which were kept in saline over ice duringthe implantation surgery (4 hours).

Protocol for the intracranial efficacy study. Two separate experimentsexamining the efficacy of the paclitaxel-polymer implant against theintracranial 9L glioma were performed. Based on in vitro clonogenicassays, the 9L glioma appears relatively resistant to paclitaxelcompared to human glioma cells. Intracranial tumor implantation in therat was performed according to the technique described by Tamargo et al.(1993), the teachings of which are incorporated herein. Briefly, a burrhole was drilled in the dura incised as described above. The cortex andwhite matter were resected with suction until the superior aspect of thebrainstem was visualized. The wound was packed with sterile gauze for 10minutes to control any bleeding. The gauze was then removed, and a 1-mm³piece of the 9L gliosarcoma was introduced into the cranial defect andplaced on the brainstem. The wound was irrigated and closed with woundclips. Surgery to implant the polymer-chemotherapy device was performedfive days later. The rats were randomized to one of the treatment orcontrol groups and weighed. The original incision was reopenedaseptically and the placement of the tumor was confirmed. A cruciateincision was made in the surface of the tumor and the polymer discadvanced into the tumor. Treatment rats received 10 mg PCPP-SA discscontaining 20, 30, or 40% paclitaxel by weight, while control ratsreceived blank 10 mg PCPP-SA discs containing no paclitaxel. Tamargo etal. have demonstrated that there is no survival difference between ratswith intracranial 9L gliomas treated with the blank PCPP-SA discs andrats given a "sham" operation without any implant. Any bleeding wasallowed to subside spontaneously, and the wound was irrigated with 0.9%saline and closed with surgical staples. The rats were examined twicedaily and the time to death recorded. Long term survivors weresacrificed either 120 days (Experiment 1) or 1 year (Experiment 2) afterimplant. At death, the brain was removed and fixed in formalin. Acoronal section was taken through the polymer implant site and stainedwith hematoxylin and eosin. The section was examined to confirm thepresence or absence of tumor growth. Survival was plotted on aKaplan-Meier survival curve and statistical significance was determinedby a nonparametric Kruskal-Wallis analysis of variance followed by anonparametric studentized Newman-Keuls test for multiple comparisons, asdescribed by Zar, J. M., Biostatistical Analysis, Prentice-Hall, Inc.,Englewood Cliffs, N.J. (1984), the teachings of which are incorporatedherein by reference.

The results of the intracranial efficacy studies are shown in Table 5.

                                      TABLE 5                                     __________________________________________________________________________    Efficacy of intracranial paclitaxel implants versus rat 9L gliosarcoma.                          Median    No. of                                                         No. of                                                                             survival                                                                          % increase                                                                          long term                                                                          P                                           Experiment                                                                           Treatment                                                                            rats (days)                                                                            life span                                                                           survivors*                                                                         value                                       __________________________________________________________________________    1      Control                                                                              8    19.5      0                                                       40% Taxol ™                                                                       6    38.0                                                                              95    2    <0.02                                              30% Taxol ™                                                                       8    45.0                                                                              131   1    <0.002                                             20% Taxol ™                                                                       8    61.5                                                                              215   2    <0.001                                      2      Control                                                                              7    18.0      0                                                       40% Taxol ™                                                                       8    35.5                                                                              97    2    <0.005                                             30% Taxol ™                                                                       8    27.0                                                                              50    0    <0.05                                       __________________________________________________________________________     *Long term survivors were alive at 120 days postimplant.                      Results of nonparametric NewmanKeuls test comparing treatment groups to       control.                                                                      The generic name for Taxol ™ is paclitaxel.                           

As shown in Table 5, two separate experiments established that thepaclitaxel polymer implant significantly extended survival in ratsbearing the intracranial 9L gliosarcoma compared to control animals.Survival was extended from 1.5 to 3.2-fold (P values from <0.05 to<0.001, respectively, nonparametric Newman-Keuls test). Each polymerpreparation produced several long term survivors (120 days or longerfrom tumor implant). The two long term survivors from Experiment 2 wereallowed to live for 1 year prior to sacrifice. None of the survivinganimals had visible tumor on autopsy either grossly or microscopicallyin hematoxylin and eosin-stained sections. In contrast, all animals inthe control groups died with large intracranial tumors. There was nosignificant survival difference among the treatment doses of eachexperiment (P>0.05, nonparametric Newman-Keuls).

In addition, two animals (20% and 30% loaded polymer groups) inExperiment 1 died during the experimental period without macro- ormicroscopic evidence of tumor growth. Scattered foci of atypical andkaryorrhectic cells similar to those seen in the rats receiving thepaclitaxel implant without tumor were present in these brains as well asin the brains of rats surviving to the end of the experimental period.The Kaplan-Meier curve for Experiment 1 is shown in FIG. 3.

Thus, the paclitaxel-polymer devices extended the median survival ofrats bearing intracranial tumors 1.5- to 3.0-fold (P <0.05 to <0.001)compared to controls.

EXAMPLE 7

Demonstration of the release kinetics of camptothecin from abiocompatible polymer.

Polymer preparation. EVAc (40% vinyl acetate by weight; Elvax 40P) wasobtained from Dupont (Wilmington, Del.). The EVAC was washed in absoluteethyl alcohol to extract the inflammatory antioxidantbutylhydroxytoluene, as described by Langer, R., et al., J. Biomed.Mater. Res. 15:267-277 (1981), the teachings of which are incorporatedherein by reference. Sodium camptothecin, obtained from the NationalCancer Institute, was incorporated into the polymer matrix by amodification of the procedure described by Rhine, W. D., et al., J.Pharm. Sci. 69:265-270 (1980), the teachings of which are incorporatedherein by reference. Camptothecin and EVAc were combined to yield 20%,40%, or 50% loaded polymers by weight. Methylene chloride was added tothe mixture to yield a 10% solution of EVAc and methylene chloride andagitated on a Vortex mixer until completely dissolved. The solution ofcamptothecin-EVAc-methylene chloride was then poured into a glass moldat -70° C. After 20 minutes, the solidified polymers were transferred toa -30° C. freezer for 4 days. The polymers were then placed in a vacuumdesiccator for 4 days at room temperature to facilitate evaporation ofmethylene chloride, after which they were stored at 4° C.

Protocol. EVAc polymers loaded with camptothecin were placed in 3.0 mlof 0.9% NaCl in a 37° C. incubator. The solution was removed at varioustime points and replaced with fresh 0.9% NaCl, thus maintaining theconcentration of camptothecin in the release medium at infinite sinkconditions. The amount of camptothecin released into the solution wasmeasured by HPLC, as described below. The cumulative dose released wasdetermined by combining the release values at each time point.

HPLC method for measuring camptothecin. Quantitative analysis wasperformed on a Beckman chromatographic system equipped with a 507Autosampler, 126AA solvent module, 166 Detector, and a System Gold datasystem. The column was a reverse-phase microbondpak C18 Waters column(particle size 10 μm, 3.9×300 mm), which was protected by an UptightPrecolumn (Upchurch Scientific, Inc.). The HPLC system was elutedisocratically with methanol:water (63:37; v/v) at room temperature. Theflow rate of the mobile phase was 1.0 ml/minute, and samples weremeasured at a wavelength of 254 nm. A standard curve was constructed byplotting peak area against concentration.

Results. EVAc polymers were prepared with loadings of 20%, 40%, and 50%camptothecin by weight. The average polymer weight was 10 mg. Thus, thetotal drug loads were approximately 2 mg, 4 mg, and 5 mg, respectively.The results of the release kinetic studies are shown in FIG. 4. With 50percent loading, an initial burst of camptothecin was released from thepolymer and steady state release was attained by 3 days, lasting atleast 21 days. The 20% and 40% loadings yielded less camptothecin ateach time point. The 50% loaded polymer was therefore selected forevaluation of efficacy in vivo based on these properties.

EXAMPLE 8

Demonstration of the efficacy of camptothecin in treating gliosarcomacells in vitro.

Tumor cell lines. The 9L gliosarcoma cells were obtained in 1985 fromDr. Marvin Barker of the University of California, San Francisco, Calif.The P98 glioma cells were provided by Dr. Joseph Goodman, Department ofNeurosurgery, Ohio State University, Columbus, Ohio. The human gliomacell lines U87 and U373 were provided by Dr. 0. Michael Colvin, JohnsHopkins University School of Medicine, Baltimore, Md. JH1 is a cell lineestablished from a biopsy specimen from a patient with a pathologicallyconfirmed glioblastoma multiform.

Cell culture of freshly biopsied gliomas. Biopsy samples were obtainedfrom the operating room and transported in sterile specimen containers.Specimens were filtered through a 230 μm mesh cellector screen (BellcoGlass, Inc., Vineland, N.J.) with use of a glass pestle. The specimenwas then centrifuged at 1000 rpm for 10 minutes. The supernatantfraction was discarded and the cell pellet was resuspended in 1 ml ofminimum essential medium (Gibco BRL, Grand Island, N.Y.) with 10% fetalbovine serum, 0.5% L-glutamine, penicillin (base; 80.5 units/ml), andstreptomycin (80.5 μg/ml). The suspension was passed throughprogressively smaller needles until it passed easily through a 25 gaugeneedle, after which it was recentrifuged at 1000 rpm for 5 minutes. Thesupernatant fraction was discarded and the pellet was resuspended in 5ml of medium. This suspension was plated onto T75 culture flasks atvarious dilutions and incubated at 37° C. The medium was changedapproximately every 3 days. When the initial culture reached confluence,the cells were trypsinized and passaged. Sensitivity was assessed by theclonogenic assay below, beginning with the second passage.

Clonogenic assay. The sensitivity of each glioma cell line was tested ina clonogenic assay. At confluence, the cells were trypsinized and platedat 400 cells per 60 mm well. After 24 hours, fresh medium containingcamptothecin at various concentrations was added. For brief exposureexperiments, the camptothecin was removed and replaced with fresh mediumafter 1 hour; for continuous exposure, medium was left in place for 7days. At 7 days, all plates were fixed and stained with coomassiebrilliant blue (Bio Rad, Richmond, Calif.). Colonies containing morethan 50 cells were identified and counted. Treatments were performed intriplicate. Survival was calculated as the number of colonies formed bythe treated cells relative to the number of colonies formed by theuntreated cells.

Results. The results of the exposure of gliomas to camptothecin in cellculture are presented in Table 6.

                  TABLE 6                                                         ______________________________________                                        In vitro sensitivity of gliomas to camptothecin                                           LD90 1 Hour                                                                              LD90 Continuous                                        Cell Line.sup.a                                                                           Exposure (μM)                                                                         Exposure (μM)                                       ______________________________________                                        F98         1.40       0.10                                                   9L          1.40       0.10                                                   U87         1.20       0.10                                                   U373        0.30       0.026                                                  JH1         0.10       0.026                                                  ______________________________________                                         .sup.aF98 and 9L are rat glioma lines. U87 and U373 are established human     glioma lines. JHI is a glioblastoma line established at Johns Hopkins         Hospital directly from a biopsy specimen. All were tested for sensitivity     to camptothecin in a clonogenic assay.                                   

The experiment was designed to assess the sensitivity of gliomas invitro to a brief (one-hour) exposure and a continuous (seven-day)exposure. When exposed for 1 hour, the LD90 ranged from 1.4 μM for 9L,F98, and U87 to 0.3 μM for U373 cells. For all the cell lines,continuous exposure for 7 days decreased the LD90 by 10- to 100-fold.For the rat and established human cell lines, the LD90 after continuousexposure was approximately 0.1 μM or less. The human glioma line JH1established directly from tumor obtained in the operating room showedthe highest sensitivity to camptothecin at both exposure times. Totalcell kill for JH1 was achieved at a concentration of 0.14 μM after a1-hour exposure and 0.03 μM after continuous exposure.

EXAMPLE 9

Demonstration of the efficacy of camptothecin in treating gliosarcoma invivo.

Animals. Male Fischer 344 rats weighing 200-250 g were obtained fromHarlan Sprague Dawley, Inc. (Indianapolis, Ind.). The animals were keptin standard animal facilities and given free access to Certified RodentChow No. 5002 (Ralston Purina Co., St. Louis, Mo.) and to Baltimore citywater.

Gliosarcoma 9L Intracranial model. The 9L gliosarcoma was maintained inthe flanks of male Fischer 344 rats. For intracranial implantation, thetumor was surgically excised from the carrier animal and cut into 1×2×2mm pieces. The pieces were kept in sterile 0.9% NaCl on ice during theimplantation procedure.

Male Fischer 344 rats were anesthetized with an intraperitonealinjection of 3-5 ml/kg of a stock solution containing ketaminehydrochloride 2.5 mg/ml, xylazine 2.5 mg/ml, and 14.25% ethyl alcohol in0.9% NaCl. The surgical site was shaved and prepared with 70% ethylalcohol and Prepodyne™ solution. After a midline incision, a 3 mm burrhole centered 5 mm posterior to the coronal suture and 3 mm lateral tothe sagittal suture was made. The dura was opened and the cortex andwhite matter were resected by using gentle suction until the brainstemwas visualized. The surgical site was irrigated until clear with sterile0.9% NaCl. A single tumor piece was placed in the depths of the corticalresection. The skin was closed with surgical staples.

Protocols for systemic and local delivery of camptothecin. Animals weredivided into four treatment groups and received camptothecin either byintraperitoneal injection or polymer-mediated local delivery.Intraperitoneal injections of camptothecin were given on days 5, 6, 7,and 8 after tumor implantation at doses of 4, 10, 20, and 40 mg/kg/day.For local delivery by polymer, camptothecin was incorporated into EVAcat a dose of 50% by weight. Polymer cylinders measuring 1×3 mm werefashioned and placed under ultra violet light for sterilization for 1 to2 hours prior to implantation. The polymers weighed on average 9.2 mg,and therefore each contained 4.6 mg of camptothecin. Polymers wereplaced directly into the tumor through the original burr hole 5 daysafter tumor implantation. Control animals and those receiving systemiccamptothecin received blank intratumoral EVAc polymers of similar sizeand weight on day 5. Animals were assessed daily for signs of toxicity,especially neurological and behavioral changes. Deaths were quantifieddaily. At the time of death, the brain was removed and placed in 10%formalin for at least 1 week. The brains were prepared for hematoxylinand eosin staining. Sections through the tumor were stained to verifythe presence of tumor.

Statistics. For the animal efficacy studies, survival was plotted on aKaplan-Meier survival curve and statistical significance was determinedby the Kruskal-Wallis nonparametric analysis of variance followed by thenonparametric analogue of the Newman-Keuls multiple comparison test.

Results. Camptothecin was evaluated for its potential to prolongsurvival in the rat 9L intracranial model when administered eithersystemically or by local polymer-mediated delivery. Table 7 shows thesurvival data for the different treatment groups.

                                      TABLE 7                                     __________________________________________________________________________    Efficacy of Camptothecin Against intracranial 9L Gliosarcoma in Fischer       344 rats                                                                               Method of      Median                                                                             Long                                             Treatment                                                                              drug           Survival                                                                           Term P                                           group  N delivery                                                                             Dose    (Days)                                                                             survivors.sup.a                                                                    value.sup.b                                 __________________________________________________________________________    Control                                                                              20                                                                              *      Blank   19    0/20                                                                              --                                                          Intracranial                                                                  EVAc                                                          Systemic                                                                             20                                                                              i.p./* Camptothecin                                                                          23   0/5  NS.sup.c                                    Campto 20       20 mg/kg/day ×                                                          4 days                                                        Systemic                                                                             5 i.p./* Camptothecin                                                                          11   0/5  NS.sup.                                     Campto 40       40 mg/kg/day ×                                                          4 days                                                        Campto 17                                                                              Intratumoral                                                                         50% load                                                                              >120 10/17                                                                              <0.001                                      polymer  EVAc   Average polymer                                                        Polymer                                                                              weight = 9.2 mg                                               __________________________________________________________________________     * Denotes treatment groups that received blank intratumoral discs of EVAc     .sup.a Long term survivors lived greater than 120 days.                       .sup.b Results of nonparametric NewmanKeuls test comparing treatment          groups to control.                                                            .sup.c NS, Not significant.                                              

Camptothecin, delivered by the polymer, significantly extended survivalcompared to controls, with 59% of the animals surviving long term(greater than 120 days, P<0.001). Systemic delivery of camptothecin didnot increase survival relative to controls. Rather, at the highest dosetested, 40 mg/kg/day for 4 days, the animals died before the controls,although this result was not statistically significant. Kaplan-Meiersurvival curves are shown in FIG. 5. There were no signs of neurologicalor behavioral abnormalities noted in any of the animals.

These data show that camptothecin can be effectively utilized by localdelivery with a controlled release polymer to prolong survival in ratsimplanted intracranially with 9L gliosarcoma. Further, the data showthat local controlled drug delivery allow the clinical use of thishighly effective drug that could not be utilized systemically because ofits toxicity and narrow therapeutic window.

EXAMPLE 10

Toxicity and Efficacy of Carboplatin delivered from sustained releasepolymers.

Carboplatin (CBDCA) has shown promise as an anti-neoplastic agentagainst both primary central nervous system (CNS) tumors and severalsolid tumors which frequently metastasize to the brain. Unfortunately,CBDCA is limited in its use for tumors in CNS by systemic toxicity andpoor penetration through the blood-brain barrier. This study assessedthe toxicity and efficacy of carboplatin delivered from sustainedrelease polymers in the treatment of experimental gliomas in rodents.

MATERIALS AND METHODS

Polymer Preparation. Two separate polyanhydride polymer systems, FAD:SAand pCPP:SA, were independently tested in this study. FAD:SA polymerdiscs were obtained from Scios Nova Inc. (Baltimore, Md.) and wereprepared as described by Domb, et al., Polymer Preprints 32, 219 (1991).The PCPP:SA polymers, formulated in a 20:80 ratio, were prepared asdescribed by Chasin, et al., Biopharm. Manufact. 1, 33 (1988). CBDCA wasobtained from Bristol Meyers Pharmaceutical Company (Syracuse, N.Y.). Itwas incorporated in pCPP:SA polymer by mix melting.

Animals--Male Fischer 344 rats weighing 200-250 g were obtained fromHarlan Sprague-Dawley (Indianapolis, Ind.) and kept in accordance withthe policies of the Johns Hopkins School of Medicine Animal Care and UseCommittee.

F-98 Glioma Inoculations. 10,000 F98 glioma cells were stereotacticallyinjected in the left parietal lobe of rats as described by Judy, et al.,J. Neurosurg. 82, 103 (1995).

Polymer Implantation. 3×1 mm polymers were placed in the brain asdescribed by Judy, et al. (1995). In toxicity studies, polymer wereimplanted on the first day of the study. In efficacy trials, polymerswere implanted in the tumor beds five days after tumor injection.

Experimental Design. Three experiments were performed. The firstmeasured toxicity of carboplatin delivered from the FAD polymer. Thesecond assessed the efficacy of the highest nontoxic polymer dosesutilizing FAD:SA polymer and three systemic doses against the F-98intracranial glioma. The third compared three doses of CBDCA releasedfrom pCCP:SA polymer. In all three studies, the animals were observeddaily for signs of neurologic or systemic toxicity and for survival.Upon death of animal, the brain was immediately removed and placed inbuffered formalin for at least five days. The specimen was imbedded inparaffin, sectioned, and stained with hematoxylin and eosin forhistologic examination.

To test toxicity, five animals per group received intracranial FAD:SApolymers containing 0%, 3%, 5%, 7% and 10% loading doses of carboplatinas described above.

To test for efficacy utilizing FAD:SA polymer, animals receivedintracranial F-98 glioma. On the fifth post-operative day, animals inthe polymer arm of the protocol received FAD:SA polymers loaded with 0%,1%, 2% or 5% of carboplatin. Animals in the systemic chemotherapy armreceived blank polymer intracranially in addition to intraperitonealinjections of carboplatin once per week for 3 weeks beginning five daysafter tumor inoculation. The three systemic doses were 10, 30, and 50mg/kg/week. The number of animals in each group is listed in Table

                  TABLE 8                                                         ______________________________________                                        Survival in Rats with Intracranial F98 Glioma After Treatment With            Carboplatin Administer by FAD-SA Polymer Directly into the Tumor              Bed or by Systemic Injection.                                                           Number          Percent                                                       of      Median  surviving 40                                        Treatment Animals Survival                                                                              days or more                                                                          Statistical Analysis                        ______________________________________                                        Control   28      16 days 0%                                                  High Dose 12      19 days 0%      p < 0.001 vs control                        Systemic                                                                      (50 mg/kg/week)                                                               Low Dose  13      23 days 0%      p < 0.001 vs control                        Systemic                                                                      (10 mg/kg/week)                                                               Local Delivery                                                                          38      32 days 5%      p < 0.001 vs control                        1% Loaded                                                                     Polymer                                                                       Local Delivery                                                                          12      35.5    33%     p < 0.001 vs control                        2% Loaded         days                                                        Polymer                                                                       Medium Dose                                                                             4       36.5    25%     p = 0.001 vs control                        Systemic          days            p = .0001 vs low                            (30 mg/kg/week)                   systemic                                    Local Delivery                                                                          15      53 days 60%     p < 0.001 vs control                        5% loaded                         p = .0069 vs 2%                             Polymer                           polymer                                                                       p < 0.001 vs high                                                             systemic                                                                      p < 0.001 vs low                                                              systemic                                                                      p = 0.027 vs                                                                  medium systemic                             ______________________________________                                    

To test efficacy from PCCP:SA polymer, 32 animals underwent intracranialinjection of F-98 tumor. Five days later, the animals were randomizedinto four groups of eight. Group 1 received blank pCCP:SA polymer.Groups 2, 3, and 4 received intracranial placement of pCCP:SA polymerloaded with 2%, 5% and 10% CBDCA respectively.

Statistical analysis was performed using EGRET and SAS software.Kaplan-Meier survival curves were determined. Comparisons between groupswithin an experiment were made by means of hazard ratio analysis.

RESULTS

Toxicity. The toxicity of locally released carboplatin from FAD:SApolymer was assessed intracranially in the rat (Table 9). The FAD:SApolymer alone showed no toxicity. The 3% and 5% loadings of carboplatineach produced a single death 4 days after implant. Surviving animalswere neurologically normal and appear systemically well. Histologyrevealed fibrosis and gliosis at the site of polymer placement. Theunderlying thalamus showed some areas of degeneration on the polymerside, but not in the contralateral thalamus. Four of five animalsreceiving 7%, and all 5 of the animals receiving 10% loaded polymer diedin the first 5 days after polymer placement. The 5% loaded polymer wasthe highest tolerated loading dose (80% long-term survival), and forthis reason, 5% loading of CBDCA in FAD:SA was chosen as the highestdose for the efficacy studies.

                  TABLE 9                                                         ______________________________________                                        Toxicity of Intracranial Carboplatin Delivered by FAD:SA Sustained            Release Polymers.                                                             Loading Dose of       Percent                                                 CBCDP in FAD:SA                                                                          Deaths in the                                                                            Surviving more                                          Polymer    First 10 days                                                                            than 120 days                                                                             Death Dates                                 ______________________________________                                        0% loading 0%         100%        no deaths                                   3% loading 20%        80%         day 4                                       5% loading 20%        80%         day 4                                       7% loading 80%        20%         day 3 (2                                                                      animals)                                                                      day 4 (2                                                                      animals)                                    10% loading                                                                              100%       0%          day 5 (5                                                                      animals)                                    ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        Survival in Rats with Intracranial Glioma After Treatment with                Carboplatin Locally Administer by pCPP:SA Polymer Release into                the Brain                                                                               Number of             Percent surviving                             Treatment Animals   Median Survival                                                                           40 days or more                               ______________________________________                                        10% Loaded                                                                              8         8 days      12.5%                                         Polymer                                                                       Control (blank                                                                          8         23 days     0%                                            polymer)                                                                      2% loaded 8         46.5 days   75% p = 0.01                                  Polymer                         vs control                                    5% loaded 8         86.5 days   75% p = 0.004                                 Polymer                         vs control                                    ______________________________________                                         Efficacy of carboplatin released from FAD:SA polymer or systemic              carboplatin treating experimental F98 glioma.                            

The intracranial F-98 glioma was uniformly fatal to untreated animals ata median of 16 days, with all animals dying by day 19. of the threeloading doses of CBDCA (1%, 2%, and 5%) tried as local therapy forintracranial F98 gliomas, 5% loading of carboplatin was the mosteffective in prolonging survival (Table 8). (Median survival 53 days.)There was early toxicity as one animal (of 15) died on each day 9 andday 10. 5% loading was statistically better than control (p<0.001) andwas also better than lower loading doses in prolonging survival(p=0.007). One percent and two percent loadings of FAD:SA alsosignificantly prolonged survival without signs of early toxicity.

Of the three systemic doses tested, the medium dose (30 mg/kg) was themost efficacious (median survival=36.5 days, p<0.001 vs. control). The5% loaded polymer was significantly better than the best systemic dosein prolonging survival in rats with F9.8 glioma (p=0.027).

Histology examination revealed that animals receiving blank polymer orineffective doses of systemic chemotherapy had large infiltrating tumorsat the site of injection. Many animals receiving the most effectivetreatment, 5% loaded FAD:SA polymer, had small or no tumor at theinjection site. Several long term survivors in the 5% local group werefree of tumor in the brain at the time of death. However, largeinfiltrative tumors of the spinal cord subarachnoid space with cordinvasion were occasionally observed in these animals.

Efficacy of CBDCA Released From pCCP:SA Polymer Against F-98 Glioma.

Median survival for animals receiving blank pCCP:SA polymer (Table 10)was 23 days. Animals receiving 2% or 5% loaded pCCP:SA polymer had asignificantly prolonged survival of 46.5 and 86.5 days respectively.Animals receiving 10%--loaded polymer had a median survival of 8 dayswith five of eight animals on day 8. This is suggestive of toxicity. Twoof eight animals receiving 5% loaded polymer died early (one each on day9 and day 10). There were no early deaths in the 2% polymer group.Except in these early deaths, autopsies revealed that animals died oflarge intracranial tumors at the site of injection of F98.

Neurotoxicity was seen in animals receiving high doses of interstitialchemotherapy of CBDCA with both polymer systems. A clear dose responsecurve was seen, with an increasing number of early deaths withescalating doses of local chemotherapy. For both polymer systems,animals receiving 10% loaded polymer died soon after implant; 5% loadedpolymer showed some early toxicity but appeared near the maximaltolerable dosing. Lower doses showed no early toxicity but wereassociated with lower efficacy.

In this study, 5% loading of CBDCA in either polyanhydride polymer wasclearly effective in prolonging survival in rats with F98 glioma. Inseveral clinical studies, human gliomas have demonstrated a mixed degreeof responsiveness to systemic carboplatin therapy. This difference inefficacy between in vitro and in vivo observations can be explained byseveral reasons including failure to adequately place the drug acrossthe blood-brain barrier into the tumor, systemic toxicity limiting thedose below the therapeutic level, and tumor cell resistance.

Interstitial chemotherapy via biodegradable polymers can increaseefficacy by addressing all three of these mechanisms. Firstly, localdelivery of platinum drugs circumvents the blood brain barrier bydelivering the chemotherapy into the tumor directly. Water solublecompounds such as the platinum drugs have relatively poor penetrationinto the CNS. As a result, their efficacy can be severely limited whenthey are delivered systemically. Secondly, local delivery can lead todecreased systemic toxicity if the blood-brain barrier (BBB) is utilizedin reverse to prevent systemic dissemination of the drug. Inexperimental studies, the maximal tolerable dose (LD₅₀) of CBDCAincreased four-fold with local delivery via polymers as compared tosystemic delivery, and CBDCA levels in the tumor were high whilesystemic levels remained low.

Conclusions Given that carboplatin has been cytotoxic to human gliomalines in vitro, has shown efficacy against CNS malignancies includingglioma in clinical trials, and has been effectively delivered frompolymer against intracranial F98 glioma in rats in this study,carboplatin delivered from polyanhydride polymer should be evaluated inhuman phase I trials. Carboplatin's delivery directly to the brain tumorsite utilizing sustained release polymers may improve its therapeuticindex and thereby increase its effect against brain tumors.

Methods: Two biodegradable polyanhydride polymer systems were loadedwith CBDCA and tested independently. Dose toxicity studies wereperformed by placement of CBDCA-loaded polymers in the brain of rats.The highest non-lethal doses were then implanted in the brains of ratsfive days after injection of F-98 glioma cells at the same site.survival of animals receiving polymer were compared with that of animalsreceiving systemic doses of CBDCA.

Results: CBDCA-polymer was well tolerated in doses up to 5% loading.Locally delivered CBDCA was effective in prolonging survival of ratswith F98 gliomas. Maximal survival was seen with 5% loading of eitherpolymer, with median survival increased two to three fold over control(p<0.004). Optimal doses of systemic CBDCA significantly prolongedsurvival. The best polymer dose was significantly more effective thanthe best systemic dose.

Conclusions: Carboplatin is effective in prolonging survival in rodentswith experimental gliomas when delivered by polyanhydride sustainedrelease polymers. Locally delivered carboplatin is more effective thansystemic carboplatin in prolonging survival in a glioma model.

Modifications and variations of the compositions of the presentinvention and methods for use will be obvious to those skilled in theart from the foregoing detailed description. Such modifications andvariations are intended to fall within the scope of the appended claims.

We claim:
 1. A chemotherapeutic composition comprisinga biocompatiblepolymeric matrix incorporating an effective amount to inhibit tumorgrowth when released in vivo at the site of the tumor of a relativelywater insoluble, relatively lipid insoluble chemotherapeutic agent,wherein the chemotherapeutic agent does not cross the blood-brainbarrier in an amount effective to inhibit growth of a solid tumor whenadministered systemically and is not paclitaxel.
 2. The composition ofclaim 1 wherein the chemotherapeutic agent is camptothecin or afunctionally effective derivative.
 3. The composition of claim 1 whereinthe polymer matrix is biodegradable.
 4. The composition of claim 2wherein the polymeric matrix is formed of a polymer selected from thegroup consisting of polyanhydrides, polyhydroxy acids, polyphosphazenes,polyorthoesters, polyesters, polyamides, polysaccharides, polyproteinsand copolymers and blends thereof.
 5. The composition of claim 1 whereinthe polymeric matrix is formed of ethylene vinyl acetate.
 6. Thecomposition of claim 1 further comprising biologically active compoundsselected from the group consisting of other chemotherapeutics,antibiotics, antivirals, antiinflammatories, targeting compounds,cytokines, immunotoxins, anti-tumor antibodies, anti-angiogenic agents,anti-edema agents, radiosensitizers, and combinations thereof.
 7. Amethod of administering to a patient in need of treatment a relativelywater insoluble, relatively lipid insoluble chemotherapeutic agentcomprisingadministering an amount of the chemotherapeutic agenteffective to inhibit growth of a solid tumor locally near or in thetumor, wherein the systemic administration of the same dosage ofchemotherapeutic agent is not effective to treat tumors and wherein thechemotherapeutic agent does not cross the blood-brain barrier in anamount effective to inhibit growth of a solid tumor when administeredsystemically and is not paclitaxel.
 8. The method of claim 7 wherein thechemotherapeutic agent is camptothecin or a functionally effectivederivative.
 9. The method of claim 7 wherein the chemotherapeutic agentis locally delivered by direct infusion to the tumor from a reservoir.10. The method of claim 7 wherein the chemotherapeutic agent is locallydelivered by implantation of a biocompatible polymer matrixincorporating the chemotherapeutic agent.
 11. The method of claim 10wherein the polymer matrix is biodegradable.
 12. The method of claim 11wherein the polymeric matrix is formed of a polymer selected from thegroup consisting of polyanhydrides, polyhydroxy acids, polyphosphazenes,polyorthoesters, polyesters, polyamides, polysaccharides, polyproteins,and copolymers and blends thereof.
 13. The method of claim 10 whereinthe polymeric matrix is formed of ethylene, vinyl acetate.
 14. Themethod of claim 7 further comprising administering radiation incombination with the composition.
 15. The method of claim 7 furthercomprising administering with the chemotherapeutic agent biologicallyactive compounds selected from the group consisting of otherchemotherapeutics, antibiotics, antivirals, antiinflammatories,targeting compounds, cytokines, immunotoxins, anti-tumor antibodies,anti-angiogenic agents, anti-edema agents, radiosensitizers, andcombinations thereof.
 16. The method of claim 7 wherein the compositionis in the form of micro-implants and are administered by injection orinfusion.